Platelet Biogenesis is developing a microfluidic bioreactor that reproduces key features of adult bone marrow (physiological microenvironment) to produce human platelets (PLTs). PLTs are the 'band- aids' of the bloodstream, responsible for clot formation and blood vessel repair. Low PLT count is a significant consequence of a radiation accident or deliberate nuclear attack, for which PLTs are a critical first-line therapy to prevent mortality due to uncontrolled bleeding. PLT units comprising ~1.5x109 PLTs/mL (3x1011 PLTs total) are currently derived exclusively from human volunteer donors, and must be stored at or above 22oC to avoid irreversible temperature-related activation/aggregation. Risk of bacterial/viral growth during room temperature storage results in short shelf life (5 days). Blood centers typically do not have more than a 1.5-day PLT inventory available for transfusion under non-emergency situations (1, 2), and there are currently no licensed PLT therapeutics for thrombocytopenia to treat radiation exposures(3). To address this major unmet need we will validate our bioreactor to generate functional PLTs from megakaryocyte progenitors at clinical scale to treat radiation-induced thrombocytopenia (RIT). To date we have shown that: (1) It is feasible to generate functional megakaryocytes and PLTs from human induced pluripotent stem cells (iPSCs, a potentially unlimited source of progenitor cells which can be stored frozen for years)(4) and (2) we can improve the rate and extent of PLT production from human iPSC-derived megakaryocytes above established static culture approaches(5). In our current bioreactor time to initiation of PLT production is reduced from 6 hours to immediately, the percent of PLT-producing progenitors is increased from 10% to more than 90%, the time to completion of PLT production (~1.5x106 PLTs per 300 L transfusion unit) is reduced from 18 hours to 8 hours, and PLT morphology, ultrastructure and in vitro function is comparable to and consistent with blood PLTs(6). Aim 1. Apply our existing bioreactor to produce 1x108 murine PLTs per 300 L transfusion unit from primary megakaryocytes, and assess PLT function in vitro. Morphology, cytoskeletal organization, granule content, ultrastructure, biomarker expression, aggregation, and clot retraction of bioreactor-derived PLTs will be compared to blood PLTs under resting and activated conditions. Aim 2. Infuse bdPLTs into sub-lethally irradiated mice to measure bdPLT immunogenicity, clearance, circulation time, hemostatic activity, and determine bdPLT function in vivo. To determine whether bdPLTs are functional in vivo, we will assess bdPLT clearance and circulation time (relative to blood PLT controls), and test their immunogenicity in genetically-distinct and isogenic mouse strains. Aim 3. Determine bdPLT hemostatic activity and thrombus formation in an isogenic adult mouse model of RIT. (non-GLP pilot study) To determine whether bdPLTs can be applied to treat RIT, we will assess bdPLT function in adult mice following sub-lethal ?-irradiation (~6.25 Gy).